A modified His-tag vector for the production of recombinant protein kinases

Histidine-tag (His-tag) is the most frequently used tag to label and purify recombinant protein kinases, namely autokinases. However, when analyzing protein phosphorylation, it appears that this modification occurs not only on the kinase itself but also on several serine residues present in the vector-derived His-tag sequence. These parasite modifications can thus lead to misinterpretation of the data concerning protein phosphorylation. We report here on a modified vector devoid of serine residues in the tag and, therefore, more appropriate and secure for studying protein phosphorylation.     

A rapid method for analyzing recombinant protein inclusion bodies by mass spectrometry

The identification and characterization of a protein overexpressed in insoluble inclusion bodies in Escherichia coli are the first crucial and time-limiting steps in recombinant protein expression. Here, a straightforward approach to the analysis of recombinant proteins in inclusion bodies is presented. Inclusion bodies were dissolved in 8M urea and analyzed by matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry without prior desalting. Mass determination was achieved by direct spotting of the samples onto the MALDI target and serial dilution in the matrix. The masses of four different proteins, expressed in inclusion bodies, were determined with a mass accuracy better than 0.1%. Furthermore, protein modifications, such as N-terminal processing of single amino acids or artificial cyanylation caused by incubation of the inclusion bodies with urea at elevated temperatures, could be detected. Similarly, tryptic digests were directly analyzed in 2M urea to obtain peptide mass fingerprints for identification and more detailed information on the primary protein structure and secondary modifications. Due to the presence of ammonia in the urea-containing buffers, no Na(+) adducts were observed in the peptide mass fingerprint analysis. Taken together, the rapid and robust procedures presented here greatly facilitate the analysis of recombinant proteins.     

Recovery of soluble human renin from inclusion bodies produced in recombinant Escherichia coli

Recombinant human renin synthesized in Escherichia coli in the form of inclusion bodies has been recovered in a soluble form without the use of denaturing agents. The renin protein in soluble fractions has been confirmed by Western immunoblotting.     

Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria

Time-dependent aggregation of a plasmid-encoded β-galactosidase fusion protein, VP1LAC, has been carefully monitored during its high-rate synthesis in Escherichia coli. Immediately after recombinant gene induction, the full-length form of the protein steadily accumulates into rapidly growing cytoplasmic inclusion bodies. Their volume increases during at least 5 h at a rate of 0.4 μm³ h⁻¹, while the average density remains constant. Protein VP1LAC accounts for about 90% of the aggregated protein throughout the building process. Minor components, such as DnaK and GroEL chaperones, have been identified in variable, but low concentrations. The homogeneous distribution of inclusion bodies among the cell population and the coexistence of large, still growing bodies with newly appearing aggregates indicate that the aggregation cores are mutually exclusive, this fact being a main determinant of the in vivo dynamics of protein aggregation.     

Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach (“InFFact”) made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli).In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E. coli lysate in most of the 12 InFFact combinations. When these proteins were fused to GFP and used in the same experiment (“InFFact-GFP”), fluorescence signals proved as sensitive and reliable as those provided by Western blotting. A trend analysis based on Western blot signals or on fluorescence allowed finding expression conditions for successfully scaling up the production of both proteins. Thus, GFP allowed InFFact trend analysis to be performed without gel electrophoresis or Western blotting.In the second part, we compared the results obtained by InFFact and InFFact-GFP when two other recombinant proteins were used which, in contrast with the proteins used in the first part, were barely detectable by Western blotting. Surprisingly, InFFact-GFP but not InFFact was able to find expression conditions for successfully scaling up the production of both proteins, suggesting that GFP could increase the solubility of the fusion partner.In conclusion, GFP allowed InFFact to be performed without gel electrophoresis and with at least the same sensitivity and specificity as that of Western blotting.     

Cloning,expression and two-step purification of recombinant His-tag enhanced green fluorescent protein over-expressed in Escherichia coli

In this report, we describe a two-step chromatographic procedure for the purification of His-tag EGFP by immobilized metal affinity expanded bed adsorption (IMAEBA) as the capture step and size exclusion chromatography as the polishing step. The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists in purifying recombinant proteins. The procedure described allowed the obtention of 230 mg pure EGFP from 1 l simple batch culture with a recovery of 90%.     

幽门螺杆菌重组HpaA蛋白包涵体的切胶纯化分析

目的 探讨切胶纯化的最佳条件及纯化重组HpaA蛋白包涵体的可行性.方法 克隆并表达pQE30-hpaA-DH5α工程菌,获得重组HpaA蛋白包涵体,使用切胶纯化法进行纯化,得到可溶性的重组HpaA蛋白.SDS-PAGE电泳后使用不同浓度、不同pH的醋酸钠溶液以及不同的染色时间染色电泳蛋白条带,分析切胶纯化的最佳条件.Western blot鉴定重组HpaA蛋白的抗原性.将重组蛋白免疫BALB/c小鼠,双向免疫扩散法检测血清抗HpaA抗体,鉴定重组蛋白的免疫原性.结果 4 mol/L醋酸钠溶液、pH 13.0、作用1 h为切胶纯化的最佳条件.经切胶纯化法获得的重组HpaA蛋白体积为6 ml,浓度为0.2942 mg/ml,蛋白量为1.7652 mg,得率为77.1%,Western blot显示该蛋白具有良好的抗原性.小鼠免疫血清能与重组HpaA蛋白反应,双扩效价大于等于1∶ 16,具有良好的免疫原性.结论 切胶能够纯化重组HpaA蛋白包涵体,获得高纯度的可溶性重组HpaA蛋白,该蛋白具有良好的抗原性和免疫原性,可用于进一步的科学研究.     

A simple strategy for the creation of a recombinant lectin microarray

Glycomics, i.e. the high-throughput analysis of carbohydrates, has yet to reach the level of ease and import of its counterparts, genomics and proteomics, due to the difficulties inherent in carbohydrate analysis. The advent of lectin microarray technology addresses many of these problems, providing a straightforward approach for glycomic analysis. However, current microarrays are limited to the available lectin set, which consists mainly of plant lectins isolated from natural sources. These lectins have inherent problems including inconsistent activity and availability. Also, many plant lectins are glycosylated, complicating glycomic evaluation of complex samples, which may contain carbohydrate-binding proteins. The creation of a recombinant, well-defined lectin set would resolve many of these issues. Herein, we describe an efficient strategy for the systematic creation of recombinant lectins for use in microarray technology. We present a small panel of simple-to-purify bacterially-derived lectins that show reliable activity and define their binding specificities by both carbohydrate microarray and ELISA. We utilize this panel to create a recombinant lectin microarray that is able to distinguish glycopatterns for both proteins and cell samples. This work opens the door to the establishment of a vast set of defined lectins via high-throughout approaches, advancing lectin microarray technology for glycomic analysis.     

N-terminal deletions and His-tag fusions dramatically affect expression of cytochrome p450 2C2 in bacteria

Mitochondria generate reactive oxygen species as by-products of oxidative metabolism. Since ascorbic acid can scavenge such destructive species, we studied the ability of mitochondria from rat liver and muscle to take up, recycle, and oxidize ascorbate. Freshly prepared mitochondria contain ascorbate, as do mitoplasts that lack the outer mitochondrial membrane. Both mitochondria and mitoplasts rapidly take up oxidized ascorbate as dehydroascorbic acid and reduce it to ascorbate. Ascorbate concentrations in mitochondria and mitoplasts rise into the low millimolar range during dehydroascorbic acid uptake, although uptake and reduction is opposed by ascorbate efflux. Mitochondrial dehydroascorbic acid reduction depends mainly on GSH, but mitochondrial thioredoxin reductase may also contribute. Reactive oxygen species generated within mitochondria oxidize ascorbate more readily than they do GSH and alpha-tocopherol. These results show that mitochondria can recycle ascorbate, which in turn might help to prevent deleterious effects of oxidant stress in the organelle.     

A high performance bench scale process for isolation from inclusion bodies,refolding and dimerisation of a thiol-engineered recombinant therapeutic protein

The use of laboratory procedures is often inefficient for materialisation of recombinant therapeutic proteins in Escherichia coli (E. coli) for pre-clinical evaluation. Approaches such as scaling out shake flask cultivation can be laborious, inefficient and expensive. These inefficiencies can be compounded if the protein requires post-translational modification such as multimerisation. We previously used laboratory methods to produce the < 60 kDa, recombinant biotherapeutic, RB1. We were aware, a priori, that dimerisation of RB1 could double the molecular weight of the protein and increase its systemic retention in the human body by avoiding renal filtration. Here we modified RB1 by substituting a native residue for an unpaired cysteine, generating eRB1, in order to favour its dimerisation. Laboratory methods failed to achieve > 20% disulphide-bridged homodimerisation or monomer of sufficient purity to enable chemi-dimerisation. As such we established a set of high performance, bench-scale, unit operations for cultivation of E. coli cells expressing eRB1, the isolation of eRB1 inclusion bodies, refolding and disulphide-based dimerisation of ≥ 40% of total eRB1 and finally successful chemi-dimerisation of remaining monomeric eRB1. The establishment of scalable procedures can now enable future investigations of eRB1 and other < 60 kDa biologics for which significant bench-scale production is required for pre-clinical evaluation.     

A simple method for histochemical demonstration of viral inclusion bodies in cell monolayers in microtitration plates

The present paper describes a Bouin's-formalin fixation and Giemsa staining procedure for demonstrating viral cytoplasmic inclusion bodies in cellular monolayers in microtitration plates. Monolayers are fixed in Bouin's fixative for 15 min followed by 10% neutral buffered formalin fixation overnight. After fixation, the monolayers are stained with 4% (v/v) Giemsa stain. The method is superior to the separate use of formalin, methanol or Bouin's fixation-staining methods and compares favorably to immunocytochemical techniques for sensitivity.     

Purification effect of artificial chaperone in the refolding of recombinant ribonuclease A from inclusion bodies

Artificial chaperone (AC) containing cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD) has been used to refold recombinant ribonuclease A (RNase A) from inclusion bodies (IBs). At low urea concentration (0.8 M), the AC could enhance the refolding yield of RNase A by effectively suppressing its intermolecular interaction-induced aggregation. As a result, 0.9 mg/mL RNase A could be 77% refolded, which was a 57% increase as compared to that without the AC. At high protein concentration range (0.9–2.3 mg/mL in total protein concentrations) and 1.6 M urea, CTAB selectively precipitated contaminant proteins distinctly, so a purification effect was achieved. For example, 1.5 mg/mL RNase A could be 62% refolded and recovered at a purity of 87%, which was a 34% increase in purity as compared to that in IBs (65%). The precipitation selectivity was considered due to the differences in the hydrophobicity of the proteins. The work indicates that by using the AC, RNase A could be efficiently refolded at low urea concentration and purified at high urea concentration from IBs at high protein concentrations.     

Penicillin-binding protein 2a of Streptococcus pneumoniae: expression in Escherichia coli and purification and refolding of inclusion bodies into a soluble and enzymatically active enzyme.

Penicillin-binding proteins (PBPs), targets of beta-lactam antibiotics, are membrane-bound enzymes essential for the biosynthesis of the bacterial cell wall. PBPs possess transpeptidase and transglycosylase activities responsible for the final steps of the bacterial cell wall cross-linking and polymerization, respectively. To facilitate our structural studies of PBPs, we constructed a 5'-truncated version (lacking bp from 1 to 231 encoding the N-terminal part of the protein including the transmembrane domain) of the pbp2a gene of Streptococcus pneumoniae and expressed the truncated gene product as a GST fusion protein in Escherichia coli. This GST fusion form of PBP2a, designated GST-PBP2a*, was expressed almost exclusively as inclusion bodies. Using a combination of high- and low-speed centrifugation, large amounts of purified inclusion bodies were obtained. These purified inclusion bodies were refolded into a soluble and enzymatically active enzyme using a single-step refolding method consisting of solubilization of the inclusion bodies with urea and direct dialysis of the solubilized preparations. Using these purification and refolding methods, approximately 37 mg of soluble GST-PBP2a* protein was obtained from 1 liter of culture. The identity of this refolded PBP2a* protein was confirmed by N-terminal sequencing. The refolded PBP2a*, with or without the GST-tag, was found to bind to BOCILLIN FL, a beta-lactam, and to hydrolyze S2d, an analog of the bacterial cell wall stem peptides. The S2d hydrolysis activity of PBP2a* was inhibited by penicillin G. In conclusion, using this expression system, and the purification and refolding methods, large amounts of the soluble GST-PBP2a* protein were obtained and shown to be enzymatically active.     

Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies

Eukaryotic proteins expressed inEscherichia coli often accumulate within the cell as insoluble protein aggregates or inclusion bodies. The recovery of structure and activity from inclusion bodies is a complex process, there are no general rules for efficient renaturation. Research into understanding how proteins fold in vivo is giving rise to potentially new refolding methods, for example, using molecular chaperones. In this article we review what is understood about the main three classes of chaperone: the Stress 60, Stress 70, and Stress 90 proteins. We also give an overview of current process strategies for renaturing inclusion bodies, and report the use of novel developments that have enhanced refolding yields.     

High-level soluble expression of hIGF-1 fusion protein in recombinant Escherichia coli

Human insulin-like growth factor 1 (hIGF-1) is one kind of growth factor with clinical significance in medicine. The expression of TrxA-hIGF-1 fusion protein was rationally compared in three different Escherichia coli hosts (BL21 (DE3), Rosetta (DE3) and Rosetta-gami (DE3)) with the transformation of plasmid pET32-hIGF-1. The highest productivity of soluble hIGF-1 fusion protein was achieved in E. coli Rosetta-gami (DE3). Moreover, the effects of different expression conditions in this E. coli Rosetta-gami (DE3)/pET32-hIGF-1 host were systematically investigated to improve the expression level of the fusion protein. Under the optimized conditions, a high percent of the target fusion protein (96%) was expressed as soluble form with the volumetric production of soluble fusion protein reaching up to 2.06 g/L. After cell disruption, the soluble fusion protein was separated effectively by affinity chromatography and cleaved by enterokinase, with the concentration of mature hIGF-1 reaching up to 0.42 g/L in the mixture. The present work should be useful for the enhanced production of soluble protein with multiple disulfide bonds in E. coli.     

A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza A virus Hemagglutinin protein

The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.